This invention relates to xcex2-amyloid (Axcex2) polypeptides, and more particularly to methods of reducing Axcex2 polypeptides using an extract, or active fraction thereof, of Cimicifuga.
Patients with Alzheimer""s disease (AD) are typically presented to a clinician by a relative who has observed a decline in memory with or without a change in other cognitive abilities such as declines in executive function, difficulty in word finding or visuo-spatial impairment such as an inability to draw complex geometric structures or becoming lost in familiar places. Neuropathological analysis of brains from patients with AD has revealed extensive neuronal and synaptic loss in select brain regions, neurofibrillary tangles, and the deposition of xcex2-amyloid (Axcex2) polypeptides in the form of senile plaques throughout the hippocampus and neocortex.
Axcex2 polypeptides are produced from the amyloid precursor protein (APP) through the combined proteolytic actions of xcex2- and xcex3-secretases and are then secreted into the extracellular milieu. Biochemical and immunocytochemical studies have revealed that the Axcex2 polypeptides deposited in brains from patients with AD have substantial amino- and carboxyl-terminal heterogeneity and can contain from 39 to 43 amino acid residues, with xcex2-amyloid 1-40 (Axcex240) and 1-42 (Axcex242) being the most predominant. By comparing signals obtained using antibodies directed at the amino terminus of Axcex2 with those obtained by capturing the peptide from an internal epitope, it was found that virtually all Axcex242 in AD brain is amino-terminally modified and/or truncated.
The invention features methods of reducing the level of an Axcex2 polypeptide using a Cimicifuga extract. The invention also features methods of producing an active fraction from an extract of Cimicifuga. An active fraction is one that is able to reduce the level of an Axcex2 polypeptide in or secreted from a cell (in vitro or in vivo). The invention further features a composition containing an active fraction of a Cimicifuga extract and an article of manufacture containing such a composition.
In one aspect, the invention provides methods of reducing the level of a xcex2-amyloid (Axcex2) polypeptide in or secreted from a cell. The methods include contacting the cell with an amount of a Cimicifuga extract or of an active fraction thereof in an amount that is effective for reducing the level of the Axcex2 polypeptide and monitoring the level of the Axcex2 polypeptide in or secreted from the cell. Representative cells include H4 cells, M17 cells, 293 cells, Chinese hamster ovary (CHO) cells, primary fibroblasts, C6, primary neuronal, primary mixed brain cultures, Daoy, SK-N-SH, SK-N-AS and SK-N-FI.
In another aspect, the invention provides methods for reducing the level of an Axcex2 polypeptide in a mammal, including administering an amount of a Cimicifuga extract or an active fraction thereof to the mammal in an amount that is effective for reducing the level of the Axcex2 polypeptide and monitoring the level of the Axcex2 polypeptide in the mammal.
In another aspect of the invention, there are provided methods of treating a mammal having AD or at risk to develop AD. The methods include administering an amount of a Cimicifuga extract or an active fraction thereof to the mammal in an amount that is effective for treating or preventing AD. Generally, the extract or active fraction thereof is administered to a mammal orally, intravenously, intracranially, intracerebrally, subcutaneously, intramuscularly, intranasally or intraperitoneally. A representative mammal is a rodent, for example, a mouse. In one embodiment, the mouse expresses an APP carrying a Swedish mutation. An example of such a mouse is a Tg2576 mouse.
The Cimicifuga extract or active fraction thereof can be from C. racemosa, and further can be obtained from the root or rhizome of a C. racemosa plant. The C. racemosa extract is generally an ethanolic or an aqueous extract. An active component within an active fraction can be soluble in a solvent such as methylene dichloride, ethyl acetate and n-butanol, and active components soluble in such solvents are typically lipophilic. An active component within an active fraction that is capable of reducing the level of an Axcex2 polypeptide can have a molecular weight of less than 10 kD.
A reduction in the level of an Axcex2 polypeptide can be due to decreased production of the Axcex2 polypeptide or increased catabolism of the Axcex2 polypeptide. In an embodiment of the invention, the level of the Axcex2 polypeptide is reduced by at least 10%, at least 25%, at least 50%, or at least 80% compared to the level of the Axcex2 polypeptide in or secreted from a corresponding cell not contacted with the extract or active fraction thereof. A reduction in the level of an Axcex2 polypeptide can be a reduction in the level of Axcex240 or Axcex242. Further, the reduction in the level of an Axcex2 polypeptide can be a preferential reduction in the level of Axcex242. In addition, the extract or active fraction thereof typically should have no significant effect on the level of one or more of APP, CTFxcex1, CTFxcex2, or sAPPxcex1.
In yet another aspect of the invention, there are provided methods of producing an active fraction of a Cimicifuga extract that reduces the level of an Axcex2 polypeptide upon contact with a cell. The methods include obtaining an extract of Cimicifuga plant material, size-fractionating the extract through a filter to obtain an active fraction, and testing the active fraction to confirm that the active fraction reduces the level of an Axcex2 polypeptide. Generally, an active fraction contains active components having a molecular weight of less than about 10 kD.
The invention also provides methods of producing an active fraction of a Cimicifuga extract that reduces the level of an Axcex2 polypeptide upon contact with a cell, including obtaining an extract of Cimicifuga plant material, extracting the Cimicifuga extract with hexane (thereby producing a hexane-soluble fraction and a hexane-insoluble fraction) and testing the hexane-insoluble fraction to confirm that the hexane-insoluble fraction reduces the level of the Axcex2 polypeptide. A representative hexane is n-hexane. Such methods can additionally include extracting the hexane-insoluble fraction with a dichloroalkane (producing a dichloroalkane-soluble fraction and a dichloroalkane-insoluble fraction) where the dichloroalkane-soluble fraction typically reduces the level of the Axcex2 polypeptide upon contact with a cell. A representative dichloroalkane is methylene dichloride. Methods of producing an active fraction also can include extracting the dichloroalkane-soluble fraction with an alkylacetate (producing an alkylacetate-soluble fraction and an alkylacetate-insoluble fraction) where the alkylacetate-soluble fraction typically reduces the level of an Axcex2 polypeptide. A representative alkylacetate is ethyl acetate. Further, methods of the invention can include extracting the alkylacetate-soluble fraction with an alcohol (producing an alcohol-soluble fraction and an alcohol-insoluble fraction) wherein the alcohol-soluble fraction typically reduces the level of the Axcex2 polypeptide. A representative alcohol is n-butanol. The product of any of the above-described extractions or fractionations can be further concentrated, for example, by lyophilizing.
In another aspect of the invention, there is provided a composition containing an active fraction of a Cimicifuga extract and a pharmaceutically acceptable carrier. Such an active fraction can reduce the level of an Axcex2 polypeptide upon contact with a cell producing the Axcex2 polypeptide.
In still yet another aspect of the invention, there is provided an article of manufacture containing an active fraction of a Cimicifuga extract, a pharmaceutically acceptable carrier, and packaging material. Generally, packaging material contains a label or package insert indicating that the composition is effective for reducing the level of an Axcex2 polypeptide.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims.
The invention provides methods of reducing the level of an Axcex2 polypeptide in or secreted from a cell. Methods of the invention include contacting a cell with an extract of Cimicifuga or an active fraction thereof in an effective amount such that Axcex2 is reduced, and subsequently monitoring the level of Axcex2.
Traditional Chinese medicine has long employed Cimicifuga species for the treatment of menstrual cramps, fatigue, anxiety, rheumatoid arthritis, alleviation of fever, pain, inflammation, sedation, swelling of joints, respiratory congestion from colds, and high blood pressure. More than two centuries ago, Native Americans were using the root of Cimicifuga racemosa, also known as black cohosh, black snakeroot, bugbane, bugwort, and squawroot, to relieve many of the symptoms associated with menstruation and menopause, including cramps, hot flashes, headaches, irritability, sweats, as well as many of the symptoms unrelated to menstruation discussed above. Cimicifuga, a perennial native woodland plant, includes several commonly known species that are used medicinally and/or ornamentally (e.g., C. simplex, C. dahurica, C. foetida, C. japonica, C. acerina, and C. racemosa). Extracts or active fractions of C. racemosa are particularly useful in the methods of the invention.
Cimicifuga Extracts and Reduction of Axcex2 Polypeptide Levels
The invention features methods of reducing the level of an Axcex2 polypeptide in or secreted from a cell. As used herein, an Axcex2 polypeptide refers to a portion of an APP (e.g., human APP) that is produced following cleavage by xcex2-secretase and xcex3-secretase (for example, residues 671-711 (Axcex240) or 671-713 (Axcex242) of GenBank Accession No. D87675). An Axcex2 polypeptide can have from 39 to 43 amino acid residues (e.g., 39, 40, 41, 42 or 43 residues). Given the heterogeneity in the length of Axcex2 polypeptides, it is a further feature of the invention that an extract can reduce the level of a specific Axcex2 polypeptide (e.g., Axcex240 or Axcex242). The ability to distinguish a reduction in the level of, for example, Axcex240 or Axcex242 is significant, since Axcex242 is considered to be more amyloidogenic. For example, immunocytochemical analysis has shown immunolabeling for Axcex242 in all types of senile plaques, poor labeling of plaques using Axcex240 end-specific antibodies, and detection of both the Axcex240 and Axcex242 epitopes in cerebrovascular amyloid. Moreover, analysis of familial AD (FAD)-linked mutations has shown elevations in extracellular Axcex2 accumulation, particularly Axcex242. As described herein, the reduction of Axcex2 polypeptides by Cimicifuga extracts can be preferential for Axcex242. As used herein xe2x80x9cpreferentialxe2x80x9d refers to a significant reduction of one Axcex2 polypeptide (e.g., Axcex242) compared to the level of one or more different Axcex2 polypeptides (e.g., Axcex240). xe2x80x9cSignificantxe2x80x9d refers to a statistical calculation of significance in which a p-value of less than 0.05 (e.g., a p-value of less than 0.025 or less than 0.01) is obtained using an appropriate statistical calculation, e.g., a paired t-test. Therefore, a p-value of greater than 0.05 indicates a lack of statistical significance based on such a calculation.
Methods of the invention include contacting a cell (in vitro or in vivo) with an effective amount of a Cimicifuga extract or active fraction thereof and monitoring the level of Axcex2 in or secreted from the cell. An effective amount of an extract or an active fraction is an amount that reduces the level of an Axcex2 polypeptide in or secreted from a cell by at least 10% (e.g., at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or completely abolishes the level of an Axcex2 polypeptide compared to the level of the Axcex2 polypeptide in or secreted from a corresponding cell not treated with the extract or active fraction. A component in an active fraction that reduces the level of an Axcex2 polypeptide can exert an effect at any of a number of steps along the pathway of Axcex2 secretion and, in the case of AD, deposition in the brain. The level of Axcex2 polypeptide not only depends upon its production, but also on the mechanisms responsible for its removal. Without being bound by a particular mechanism, a reduction in the level of an Axcex2 polypeptide can be due to the activity of binding proteins that sequester the polypeptide, or to other cellular mechanisms such as decreased production or increased catabolism of an Axcex2 polypeptide. By way of example, catabolism of an Axcex2 polypeptide likely involves both intracellular (e.g., acting at the site of Axcex2 polypeptide generation and/or within the secretory pathway) and extracellular (e.g., cell-surface, secreted, endosomal and/or lysosomal) proteases. Compounds that reduce the level of one or more Axcex2 polypeptides can be useful, for example, as therapeutic compounds or to design therapeutic compounds.
Cells can be contacted in vitro with a Cimicifuga extract or active fraction thereof while in cell culture (e.g., introducing an extract into culture media). Representative cells that can be used include, but are not limited to, H4 neuroglioma, M17 neuroblastoma, human kidney 293, Chinese hamster ovary (CHO), primary fibroblast, C6, primary neuronal, primary mixed brain, Daoy, SK-N-SH, SK-N-AS and SK-N-FI-cells. Cells suitable for primary screening can be obtained from the American Type Culture Collection (ATCC) (10801 University Blvd., Manassas, Va. 20110). In addition, such cells can be transgenic cells carrying a construct containing a nucleic acid encoding APP or any portion of APP containing an Axcex2 fragment. For example, H4xcex2APP695wt cells are H4 cells carrying a human wild-type 695-amino acid APP isoform. Isoforms of APP that are produced by alternate mRNA splicing include the aforementioned APP695 as well as APP751 and APP770. In addition, cells can express human APP carrying a mutation that, for example, changes a lysine-methionine at amino acid residues 670 and 671 to an asparagine-leucine (i.e., the Swedish mutation) or one that changes a valine to an isoleucine at amino acid residue 717.
A Cimicifuga extract or active fraction therefrom also can be administered to a mammal such as a human or a rodent to reduce the level of Axcex2. For example, a Cimicifuga extract can be administered to an individual over the age of 60, or to an individual diagnosed with AD or that is at risk for developing AD (e.g., based upon familial history or the presence of a mutation associated with AD). A Cimicifuga extract also can be administered to a rodent such as a guinea pig or a mouse, and in particular, to an animal model of AD. Animal models of AD are available that accumulate Axcex2 and that develop plaques in an age-dependent manner. In particular, transgenic mice are available that carry a mutation in a presenilin gene and/or express an APP carrying a mutation (for example, a Swedish mutation). A representative mouse carrying a Swedish mutation is Tg2576 (Holcomb et al., 1998, Nat. Med., 4:97-100). The Tg2576 mouse accumulates Axcex2 polypeptides and develops plaques in an age-dependent manner. A Cimicifuga extract or active fraction thereof can be administered to a mammal by any route, including orally, intravenously, intracranially, intracerebrally, subcutaneously, intramuscularly, intranasally or intraperitoneally.
Since a reduction in the level of an Axcex2 polypeptide in vitro or in vivo can be a direct result of a decrease in cell viability, toxic effects of a Cimicifuga extract or active fraction thereof on a cell or a mammal are typically evaluated. Methods to evaluate cellular toxicity are known to those of skill in the art. For example, the degree of conversion of a tetrazolium salt (e.g., MTS) into formazan is directly proportional to the number of metabolically active cells on a cell culture plate. Conversion of MTS can be measured using the CellTiter 96(copyright) assay kit (Promega; Madison, Wis.). Further, the number of lysed cells is quantitatively proportional to the LDH (lactate dehydrogenase)-catalyzed conversion of tetrazolium salt to red formazan. The Cytotox 96 assay kit (Promega) can be used to examine LDH release.
To address issues of toxicity in vivo, standard haematoxylin/eosin (H/E) staining can be performed on the liver, kidney, spleen, and brain of an animal (e.g., a rodent such as an animal model of AD) to look for any abnormalities apparent between the extract-treated and control groups. In addition, serum levels of blood urea nitrogen (BUN), aspartate amino transaminase (AST-SGOT), and alkaline phosphatase (ALP) can be monitored in an animal to evaluate renal function, liver damage, and drug-induced biliary obstructions.
Methods of Detecting Axcex2 Polypeptides
Methods for detecting Axcex2 in cell culture or in a biological sample from an individual (e.g., plasma or cerebrospinal fluid) are known to those of skill in the art. High throughput screens have been developed to examine the level of Axcex240 or Axcex242 in medium conditioned by a cell line over time. A highly specific sandwich ELISA is described herein using a BAN-50 antibody, which specifically captures Axcex2 at the N-terminus, and then either a BA-27 antibody, which detects only full-length Axcex2 polypeptides ending at position 40, or a BC-05 antibody, which detects only full-length Axcex2 polypeptides ending at position 42 (Suzuki et al., 1994, Science, 264:1336-40; Asami-Odaka et al., 1995, Biochem., 34:10272-8).
Suitable antibodies that detect an epitope within Axcex2 (or within the Axcex2 portion of APP) are also commercially available from a variety of sources, including, but not limited to, Biosource International (Camarillo, Calif.), Senetek PLC (London, England), Zymed Laboratories (San Francisco, Calif.), Peninsula Laboratories (San Carlos, Calif.) and Boehringer Mannheim (Indianapolis, Ind.). Several of the commercial sources listed herein also provide antibodies with specific binding affinity for either Axcex240 or Axcex242. In addition, BNT-77 is an antibody that captures rodent Axcex2 (Asami-Odaka et al., 1995, Biochem., 34:10272-8) that can be used in conjunction with the BA-27 or BC-05 antibodies described above,
In addition to the specific sandwich ELISA described herein, other types of solid phase immunoassays as well as immunoassay formats such as Western blots or immunoprecipitations may be used to detect Axcex2 and are well known in the art. See, Short Protocols in Molecular Biology, Ch. 11, John Wiley and Sons, Ed., Ausubel et al., 1992. Solid-phase immunoassays include competition immunoassays, immobilized-antigen immunoassays, immobilized-antibody immunoassays, and double-antibody immunoassays. For example, a typical double-antibody immunoassay to detect Axcex2 can include the following steps: attaching an antibody with binding affinity for Axcex2 to a solid support; exposing the antibody to unlabeled Axcex2 polypeptide; washing to remove unbound Axcex2; and quantitating the amount of Axcex2 bound to the immobilized antibody using an excess of a second antibody. The second antibody can have binding affinity for Axcex2 and can be radiolabeled or conjugated to a chemical or enzyme for detection following addition of an appropriate substrate.
Western blotting to detect Axcex2 typically includes the steps of electrophoretically separating peptides in or secreted from a cell; transferring the peptides from the separation medium (e.g., a gel) to a solid support (e.g., nitrocellulose, nylon); and probing with antibodies with binding affinity for Axcex2. Probing can be direct (e.g., a labeled primary antibody) or indirect (e.g., an unlabeled antibody specific for Axcex2, which is subsequently detected with a labeled secondary antibody or immunological reagent, for example, protein A or anti-immunoglobulin).
Axcex2 immunoprecipitation methods generally include the following steps: radiolabeling cells expressing Axcex2; lysing the cells; forming specific immune complexes between Axcex2 and an antibody with binding affinity for Axcex2; collecting and purifying the immune complexes; and analyzing the radiolabeled Axcex2 in the immunoprecipitate. Immunoprecipitation is often used to detect and quantitate target antigens in complex mixtures of proteins. Immunoprecipitation can be used to analyze unlabeled proteins from unlabeled cells, provided sufficiently sensitive methods are available to detect the target protein after it has been dissociated from the antibody.
A detectable label, e.g., a radioactive label (e.g., 3H, 125I, 131I, 32P, 35S, and 14C)or a non-radioactive label (e.g., a fluorescent label, a chemiluminescent label, a paramagnetic label, or an enzyme label) may be attached to an antibody or a fragment thereof using techniques known to those of ordinary skill in the art. Examples of enzyme labels used routinely in the art for detection and quantitation include horseradish peroxidase (HRP) and alkaline phosphatase (AP). The substrates available for either HRP or AP labels are known in the art and can be selected based upon the desired method of detecting complex formation (e.g., a fluorogenic, chemiluminescent or calorimetric signal).
Axcex2 that has been detected by any of the methods described herein or other methods known to those of skill in the art can be visualized or quantitated using methods routine in the art, including autoradiography of a radioactive label (e.g., x-ray film, phosphorimaging, or densitometric analysis) and spectrophotometry of a fluorescent label or of a calorimetric reaction produced by, for example, an enzymatic label. In addition, a non-Axcex2 polypeptide also can be detected and quantitated using methods similar to those described herein for Axcex2 (e.g., for normalization purposes).
In addition, mass spectrometry (MS) can be used to detect and quantitate Axcex2 polypeptides. Several types of MS are available and routinely used in the art, and include Fourier-transform MS, Ion-trap MS, Magnetic-sector MS, Quadropole MS and Time-of-flight (TOF) MS. By way of example, Ciphergen (Fremont, Calif.) sells a biochip system for capturing Axcex2 polypeptides from culture medium or a biological sample and utilizes SELDI technology (Surface-Enhanced Laser Desorption/Ionization) with TOF-MS to detect and quantitate the level of Axcex2 polypeptides.
Neuritic plaques in the brain of an individual with AD can be detected and/or monitored in vivo using an Axcex2 polypeptide covalently modified with a polyamine (e.g., putrescine, spermidine, or spermine) (Wengenack et al., 2000, Nat Biotechnol., 18:868-72). A radiolabeled polyamine-modified Axcex2 polypeptide can be administered to an individual intravenously and detected using standard methods. For example, a radiolabel suitable for diagnostic imaging can be used (e.g., 123I) and detected using single photon emission computed tomography (SPECT).
Producing Cimicifuga Extracts and Active Fractions Thereof
Extraction is a process whereby the desired constituents of a plant or plant part are removed using a solvent or other means. Generally, an extract of Cimicifuga is obtained from the root or rhizome of the plant, although leaves, stems and flowers also can be used. The Cimicifuga extract or active fraction can be from C. racemosa. To produce an extract, plant material is usually first cleaned and dried if necessary. Drying can be done naturally (e.g., by air drying) or artificially (e.g., using warm-air fans or conveyor dryers). The plant material then can be ground, cut, or shredded using, for example, hammer action, pressure, friction or impact cutting. Methods of removing the desired constituents from the plant material include, but are not limited to, organic solvent extraction, supercritical gas extraction, and steam distillation. By way of example, there are a number of procedures for organic solvent extraction, including maceration (soaking and agitating the plant material with a solvent), percolation (repeated rinsing of the plant material with a solvent), and countercurrent extraction (continuous flow of a solvent in the opposite direction as the plant material). Representative solvents include, but are not limited to, ethanol, benzene, toluene and ether. Aqueous extracts, such as decoctions (boiling the plant material, generally used for hard tissues), infusions (steeping the plant material, generally used for soft tissues) or macerations, can also be produced, although microbial contamination can be a concern with aqueous extraction methods. As used herein in the methods of the invention, a C. racemosa extract can be an ethanolic extract or an aqueous extract, depending upon the solubility of an active component that reduces the level of an Axcex2 polypeptide. Extracts of Cimicifuga are commercially available, and include Remifemin(copyright) (SmithKline Beecham, Research Triangle Park, N.C.), and Black Cohosh (Viable Herbal Solutions, Morrisville, Penn.; or Whole Health Discount Center, http://www.WholeHealthDiscountCenter.com).
Methods of producing active fractions (i.e., containing one or more active components) from a Cimicifuga extract are provided by the invention. Active components of a Cimicifuga extract or active fraction can include, but are not limited to, polyphenols, flavonoids, aromatic acids, metabolites, alkaloids, proteins, carbohydrates, starches, steroids, resins, elements or combinations thereof (e.g., glycoproteins) that, alone or in combination with other components, can reduce the level of an Axcex2 polypeptide. For example, fractionating by traditional solvent extraction employs partitioning of a solute between two immiscible phases, typically an organic phase (e.g., n-hexane, methylene dichloride, ethyl acetate or n-butanol) and an aqueous phase. Rapid extraction kinetics and the ability to utilize a number of different diluents, extractants, and aqueous phases makes solvent extraction a powerful separation method. In addition, numerous other separation procedures can be employed to further purify desired components or remove unwanted or contaminating components, including decanting, filtration, sedimentation, centrifugation, heating, adsorption, precipitation, chromatography, or ion exchange. The resulting active fraction can be subsequently concentrated by evaporation, vaporization, lyophilization or vacuum drying. Although the invention exemplifies fractionating using organic solvent partitioning, those of skill in the art are aware of the advantages of using certain separation techniques in combination with others to increasingly partition one or more active components into active fractions.
One method of the invention includes obtaining an extract of Cimicifuga plant material and size-fractionating the extract, e.g., through a filter. The flow-through represents an active fraction. Using this or a similar method of fractionating a Cimicifuga extract, an active component within the active fraction can pass through a 10 kilodalton (kD) nominal molecular weight filter or concentrator.
Another method of producing an active fraction of a Cimicifuga extract includes obtaining an extract of Cimicifuga plant material, and extracting the extract with hexane (e.g., n-hexane). The hexane-insoluble fraction can be used to reduce the level of an Axcex2 polypeptide following contact with a cell or can be further fractionated. For example, the hexane-insoluble fraction can be extracted with a dichloroalkane (e.g., methylene dichloride or dichloroethane). As described herein, the dichloroalkane-soluble fraction is able to reduce levels of Axcex2. An active component within the dichloroalkane-soluble fraction can be further partitioned by extracting with an alkylacetate (e.g., ethyl acetate or methyl acetate) and obtaining an alkylacetate-soluble fraction. An active component within the alkylacetate-soluble fraction can be further partitioned by extracting with an alcohol solvent (e.g., n-butanol or 1-pentanol) and obtaining the alcohol-soluble fraction. Those of skill in the art are aware of additional steps during fractionating that can facilitate sample handling (e.g., concentrating an active fraction).
Given the high throughput cell based assays described herein, hundreds or thousands of fractions or combinations of fractions can be rapidly screened to identify those containing one or more active components or to identify fractions that, when combined, confer activity. An animal such as the Tg2576 mouse or a transgenic mouse carrying a Swedish mutation and a mutation in a presenilin sequence also can be used to evaluate the level of Axcex2 (e.g., in plasma or homogenized brain using, for example, the sandwich ELISA described herein), plaque burden (e.g., in brain tissue using, for example, immunocytochemical analysis), behavior/memory (e.g., using a Morris water maze to evaluate spatial reference memory), or other neurological markers such as differences in astroglia, dystrophic neuritis, microglia and the state of tau phosphorylation (e.g., by immunocytochemical analysis of brain tissue) following administration of a Cimicifuga extract or active fraction.
An active component from a Cimicifuga extract or active fraction that is able to reduce Axcex2 levels can be soluble in methylene dichloride, ethyl acetate and n-butanol, generally indicating a lipophilic active component. Lipophilic compounds are those that prefer a nonpolar environment to an aqueous one. Compounds from C. racemosa extract have been identified previously using methods such as those described herein, and include a phytoestrogen capable of binding estrogen receptors, and cimicifugin and macrotin resins, which effect, among others, the reproductive and nervous systems. Also identified from Cimicifuga extracts are triterpene glycosides, including cimicifugoside, which is believed to affect the hypothalamus-pituitary system and the reproductive and nervous systems, actein, a steroidal derivative that lowers blood pressure in animals, 27-deoxyactein, and racemoside. An isoflavone called formonenetin has been identified as binding to estrogen receptors in the rat uterus. Aromatic acids, including ferulic acid and isoferulic acid are believed responsible for the extract""s anti-inflammatory effects.
Compositions and Articles of Manufacture
The invention further features a composition containing an active fraction of a Cimicifuga extract. A composition containing an active fraction of a Cimicifuga extract can be in any form provided the composition can be placed in contact with a cell in an amount and for a length of time effective to reduce the level of an Axcex2 polypeptide.
Compositions of the invention may be administered on a continuous or an intermittent basis. By way of example, a composition within the scope of the invention can be in the form of a liquid, solution, suspension, pill, capsule, tablet, gelcap, powder, gel, ointment, cream, nebulae, mist, atomized vapor, or aerosol. For the purpose of this invention, routes of administration include, but are not limited to, oral, nasal, intravenous, intramuscular, intraperitoneal, subcutaneous, intrathecal, intradermal, or topical. The route of administration can depend on a variety of factors, such as the environment in which cells are contacted and therapeutic goals. The dosages of a particular composition will depend on many factors, including the mode of administration and the cells being treated. Typically, the amount of an active fraction contained within a single dose of a composition will be an amount that effectively reduces the level of an Axcex2 polypeptide without inducing significant toxicity.
In addition, compositions within the scope of the invention can contain a pharmaceutically acceptable carrier for in vivo administration to a mammal, including, without limitation, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include, without limitation, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters. Aqueous carriers include water, alcohol, saline, and buffered solutions. Pharmaceutically acceptable carriers can also include physiologically acceptable aqueous vehicles (e.g., physiological saline or artificial cerebral-spinal fluid) or other known carriers appropriate to specific routes of administration. Additional compounds can be included in a composition, such as steroids, mucolytic agents, anti-inflammatory agents, immunosuppressants, dilators, vasoconstrictors, or combinations thereof. Preservatives, flavorings, and other additives such as, for example, anti-microbials, anti-oxidants, chelating agents, inert gases, and the like may also be present in a composition.
For oral administration, tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets can be coated by methods known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspension, or can be presented as a dry product for constitution with saline or other suitable liquid vehicle before use. Liquid preparations also can contain pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles, preservatives, buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the compound.
The invention further provides for an article of manufacture that includes a composition containing an active fraction of a Cimicifuga extract, and packaging material. The packaging material in or on an article of manufacture indicates that the composition therein is effective for reducing the level of an Axcex2 polypeptide. Components and methods for producing articles of manufactures are well known in the art. Instructions, for example on a label or package insert, describing a particular dose of the composition to be administered may be included in such kits. Different kits may be manufactured that contain a composition in different forms (e.g., a pill or a liquid) or that contain a dose of a composition appropriate to reduce the level of an Axcex2 polypeptide in, for example, an adult female subject or in an adult male subject (based upon an average weight). Instructions can further include a table or chart for adjusting a particular dose of a composition for a subject that deviates from average.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.